This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. I study the intracellular diffusivity of a model substrate (NLS-GFP) by using a novel and unconventional fluorescence correlation technique, based on pair correlation functions. I detect molecular transport events by measuring the time a single molecule takes to move from a specific location to another within the cell. Here is the novelty with respect to the traditional FCS approach that measures the time a molecule takes to cross the laser beam. By this approach obstacle to diffusion can be detected both within cellular compartments (restrictions to free diffusion) and across the nuclear envelope barrier (restrictions due to the presence of nuclear pore channels). In both cases the measurement affords a quantitative description of the kinetic steps of NLS-GFP transport at the single molecule level, and in living cells, without the need for complex labeling procedures and cell manipulations.